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2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 1
Sponsored by:
RIIP
Reseau International Institut Pasteur
International Course 2016
“Cell Biology and Infection: Digital Image
Processing/Analysis Tools for Quantitative Light
Microscopy Imaging”
Hosting organization:
Hellenic Pasteur Institute Light Microscopy Unit
Sponsors:
RIIP, Institut Pasteur, Institut Carnot Pasteur MI
Hellenic Pasteur Institute
Main Organizer:
Haralabia Boleti Co-organizers:
Jean Christophe Olivo-Marin,
Evangelia Xingi & Fabrice de Chaumont
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 2
Sponsored by:
Preface
Advanced Light Microscopy is an essential tool for research and diagnosis in most areas of biomedical sciences. Direct visualization of live biological samples when combined with quantitative analysis of data extracted from digital images, can provide novel insight or reinterpretation of biological questions investigated in the past only by biochemical methods. The latest developments in Fluorescence microscopy (i.e. confocal and multiphoton microscopy, fluorescence time lapse video microscopy, techniques as FRAP, FRET, FLIM, FCS for molecular imaging and determination of cell dynamics, super resolution microscopy etc) that allow molecular 3D and 4D imaging of multiple fluorochromes simultaneously and the determination of cellular dynamics at 5D, make an absolute requirement the use of highly sophisticated computer software for 3D modeling and quantitative analysis and interpretation of the imaging data. The rapid progress in Live Cell Imaging, Bioinformatics and Digital Image analysis tools has created a gap in the training of scientists using advanced imaging approaches in Digital image processing. A real need therefore exists for continuous training of the microscopy users on the various image analysis software. This training will enable them to extract quantitative information from still images or videos from live imaging experiments and thus, fully exploit the information contained in their microscopy data to draw accurate and in depth conclusions about the biological phenomena they study. This course is addressed to scientists from all Institutes of the Institut Pasteur International Network (IPIN) but also to scientists from all academic institutions. We hope that it will serve as an “incubator” for new networking and new possibilities for collaborations. Finally, we are very happy to emphasize the real international character of this course, where students and instructors from 11 different countries and 4 continents participate.
Course description This 5-day practical course entitled “Cell Biology and Infection: Digital Image Processing/Analysis Tools for Quantitative Light Microscopy Imaging” focuses on topics related to biomedical applications in the fields of infectiology and cell biology.
Its main objective is to provide theoretical and practical training in basic and advanced concepts and methods of digital image analysis and in the use of two open source (Icy and Fiji) software for quantitative assessment of fluorescence microscopy data through:
• Lectures • Hands-on practical training with participants’ microscopy data or data provided by the
instructors • Informal discussions and tutorials with experts in the field
The course is addressed to PhD students, post doctoral fellows and young investigators who carry out research projects requiring fluorescence microscopy imaging and digital image processing.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 3
Sponsored by:
Hellenic Pasteur Institute
http://www.pasteur.gr/?lang=en
The Hellenic Pasteur Institute (HPI), a member of the Institut Pasteur International Network, is a non-profit research foundation that operates through a bilateral agreement between the Greek Government and the Institut Pasteur in Paris, France. Since its establishment in 1920, the HPI remains faithful to its mission, which is the prevention and treatment of Diseases through Basic Research, Education and Public Health Services. Education at HPI focuses on the training of young scientists (PhD, MSc and diploma students) and the dissemination of specialized scientific information to specific public target audiences. Besides the training of young researchers in the HPI laboratories, HPI researchers organize national and international scientific training courses for researchers and graduate students on cutting-edge technologies. Additionally, in the context of Science and Society activities, the HPI has organized courses on biosciences and bioethics for secondary school teachers and hosts secondary- and primary-school students’ visits for educational purposes. The Hellenic Pasteur Institute Light Microscopy Unit (HPI-LMU) (http://www.pasteur.gr/?page_id=2444&lang=en), is one of the most modern and well-equipped imaging facilities in Greece. It provides advanced light microscopy tools and services to in-house and visiting scientists and is involved in several training projects at national and international level.
RIIP (Reseau International Institut Pasteur) IPIN (Institut Pasteur International Network)
http://www.pasteur.fr/en/international
The International Network of Pasteur Institutes and Associated Institutes (Reseau International des Instituts Pasteur et Instituts Associés) comprises 33 Institutes in all continents which have signed in 2010 a new Collaboration Agreement of the Institut Pasteur International Network. Τhe Pasteur Institute in Paris has been the core for the development of this International Network.
For over a century, the contribution of the Institut Pasteur International Network essentially coincides with the history of discoveries that has marked the development of the biomedical sciences and the struggle against some of humanity’s greatest health problems: The boubonic plague by Yersin, typhoid fever by Laveran, diphtheria by Roux and Ramon, tuberculosis by Calmette and Guerrin. Also, this contribution includes some of the early steps of molecular biology by A. Lwoff, J. Monod and F. Jacob and the isolation of the AIDS virus by J. L. Montagnier. Eight Nobel prizes, eight landmarks in the history of medicine.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 4
Sponsored by:
Every year, the Institut Pasteur International Network organizes and finances teaching & training within the Network reserved for the Institutes’ researchers, the technicians or the students and for the exterior personnel (ministries, universities), who can then use their experience in the other national or regional structures.
Contact person: Eliane COËFFIER [Project Manager (Institutional relations, grants, teaching and training, communication)]
International division, 25-28 rue du Dr Roux, 75015 Paris, FRANCE E-mail: [email protected] Tel: +33(0)1 40 61 38 29
Institut Pasteur
http://www.pasteur.fr/en
Since its creation in 1887, the Institut Pasteur has become famous throughout the world as a symbol of science and French culture. For 120 years, it has been contributing to the prevention and treatment of infectious diseases through research, teaching and public health initiatives.
Across the globe, thousands of researchers, trained or working at the Institut Pasteur, share values that form the very core of the Pasteurian community: an original scientific approach, an ongoing preoccupation with applying research to public health needs, professional ethics, a desire to care for people regardless of their background or nationality, and an outward-looking attitude, sharing knowledge and expertise with the international community. These guiding principles make the Institut Pasteur a unique institution which values exchanges and partnerships. With its unique setup and prestigious history, the Institut Pasteur has always stood at the forefront of innovation, adapting to the rapidly developing world of biological research and its applications, in particular in the field of biotechnology, but most importantly to the fields of infectious diseases,
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 5
Sponsored by:
microbiology, virology and immunology.
Carnot Foundation Maladies infectieuses
http://www.instituts-carnot.eu/en/carnot-institute/pasteur-mi
The Carnot Institute "Pasteur Infectious Diseases" consists of five thematic departments of the Institut Pasteur (virology, microbiology, parasitology and mycology, infection and epidemiology, cell biology and infection), which in association with the CNRS and INSERM is dedicated to research on infectious diseases. It brings together rising and completed research activities, and makes them subject of many research partnerships with SMEs and large companies in the pharmaceutical and diagnostics. Pasteur MI strives to combine a unique know-how with multidisciplinary scientific approaches to offer its industrial partners relevant answers to major economic and public health issues related tο
• Mechanisms of bacterial, viral, fungal and parasitic pathogenesis • Genetics and predisposition to infections • Microbiota and human health • Integrative biology • Clinical and epidemiological studies • New antimicrobial strategies • Biotechnologies
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 6
Sponsored by:
“Cell Biology and Infection: Digital image processing/analysis tools for Quantitative Light Microscopy Imaging”
PROGRAM 1st Day, Monday, July 4
Processing and quantification of biological images/ tools and applications
08:30-09:00 Registration
09:00-09:30 Haralabia Boleti, Hellenic Pasteur Institute
Introduction to the course/Opening remarks
09:30-10:30 Participants
Introductory presentations on their research projects and aims related to the course
10:30 - 10:50 Coffee break
10:50 - 11:40 Jean-Christophe Olivo- Marin, Institut Pasteur
Image analysis and computer vision tools for the processing and quantification of biological images. Icy, the open source computer software for bioimaging
11:40 - 12:30 Szymon Stoma, ScopeM, ETH Zurich,
Introduction to image processing and analysis using FIJI/ImageJ
Practical session on ImageJ/Fiji
12:30 - 13:30 ImageJ/Fiji hands on practical
Instructors: S. Stoma, E. Xingi, F. de Chaumont
13:30 - 14:30 Lunch
14:30 -16:30 ImageJ/Fiji hands on practical
16:30 - 16:50 Coffee break
16:50 - 18:30 ImageJ/Fiji hands on practical
Free time
21:00-23:00 Welcoming Dinner (students and instructors)
Program
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 7
Sponsored by:
2nd Day, Tuesday, July 5
Processing and quantification of microscopy data / tools and applications
09:00 - 9:45 Dimitra Thomaidou, Hellenic Pasteur Institute
Αnalysis of digital images and videos derived from multiphoton microscopy intravital imaging
09:45 - 10:30 Fabrice de Chaumont, Institut Pasteur
Icy: new features and future
10:30 - 10:50 Coffee break
Practical session on Icy
11:00- 13:30 Icy hands on practical
Instructors: F. de Chaumont, A. Dufour, S. Dallongeville, L. Danglot, J. Mutterer
13:30- 14:30 Lunch
14:30 -16:30 Icy hands on practical
16:30-16:50 Coffee break
16:50-18:30 Icy hands on practical
3rd Day, Wednesday, July 6
Cell motility and dynamics of phagocytosis and intracellular traffic
09:00- 09:45 Alexandre Dufour, Institut Pasteur
Deciphering cellular morpho-dynamics using Bioimage Informatics
09:45-10:30 Chiara Zurzolo, Institut Pasteur
Imaging Prions spreading in neurodegenerative diseases: Role of Tunneling Nanotubes (TNTs)
10:30 - 10:50 Coffee break
10:50 - 11:35 Florence Niedergang, Institut Cochin
Imaging phagosome formation, closure and maturation in normal and virus-infected macrophages.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 8
Sponsored by:
Practical session on ImageJ/Fiji
11:40- 13:30 ImageJ/Fiji hands on practical
Instructors: S. Stoma, E. Xingi, F. Nierdengang, J. Mutterer
13:30-14:30 Lunch
14:30 - 16:30 ImageJ/Fiji hands on practical
16:30-16:50 Coffee break
16:50-18:30 ImageJ/Fiji hands on practical
4th Day, Thursday, July 7
Super-resolution and high throughput Imaging based screening
09:00- 09:45 Lyndia Danglot, Institut Jacques Monod
Morphometric analysis of neuronal networks: from memory to super-resolution structure of the synapse
09:45-10:30 Franc Perez, Institut Curie
Systematic analysis of the secretory pathway in mammalian cells
10:30 - 10:50 Coffee break
10:50 - 11:35 J. Pizarro Cerda, Institut Pasteur
Automated High Throughput Microscopy-based RNAi screening of mammalian cell invasion by the bacterial pathogen Listeria monocytogenes
Practical session on Icy
11:40- 13:30 Icy hands on practical
Instructors : F. de Chaumont, A. Dufour, S. Dallongeville, F. Perez, J. Mutterer
13:30- 14:30 Lunch
14:30 -16:30 Icy hands on practical
16:30-16:50 Coffee break
16:50-18:30 Icy hands on practical
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 9
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5th Day, Friday, July 8
Imaging of pathogen host interaction
09:00- 09:45 Freddy Frischknecht, Center for Infectious Diseases, Heidelberg University Understanding malaria parasite migration
09:45-10:30 Isabelle Tardieux, Institut of Advanced BioSciences, Univ. Grenoble Alpes
Multi-imaging approaches to decode how Toxoplasma parasites invade target cells
10:30 - 10:50 Coffee break
10:50 - 11:35 Jerome Mutterer, Institut de Biologie Moléculaire des Plantes
Image ethics for publication (what can be done and what should be avoided)
Informal tutorials with the instructors/ Practice on Icy and ImageJ/Fiji
11:40- 13:30 Instructors : F. de Chaumont, A. Dufour, S. Dallongeville, S. Stoma, J. Pizarro Cerda, E. Xingi, I, Tardieux, F. Frishchnecht, J. Mutterer
Participants will practice with the help of the instructors on their projects using tools from Icy or ImageJ/Fiji
13:30- 14:30 Lunch
14:30 -16:00 Preparation of participants' presentations
16:00-17:45 Participants' presentations on the results from their course projects
17:45-18:00 Haralabia Boleti, Hellenic Pasteur Institute
Closing remarks, END of Course
19:30- 21:00 Cultural / Social event
Guided tour to the New Acropolis Museum
21:00-23:00 Farewell Dinner
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 10
Sponsored by:
ABSTRACTS
&
OUTLINES
of
PRACTICAL TRAINING
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 11
Sponsored by:
Image analysis and computer vision tools for the processing and quantification of biological images.
Icy, the open source computer software for bioimaging
Jean Christophe Olivo Marin
Bioimage Analysis Unit, Institut Pasteur, Paris, FRANCE
An increasing number of biological projects aim at elucidating the links between biological function
and phenotype through imaging and modelling the spatiot emporal characteristics of cellular or
organism dynamics. In a firts part, we will present and discuss some recent developments of robust
and automated tools and software for flexible and robust quantitative assessment of 2D/3D+t dynamic
imaging data from biological projects. Thanks to these tools, it is possible in a large number of
experiments to automate the extraction of quantitative data from images and to facilitate the
understanding of the biological information contained therein.
Then we will present Icy, a free open-source software that provides an integrated web-based
development platform for bioimage informatics applications. Icy offers a common platform for both
image analysis scientists developing new algorithms and biologists seeking for a powerful and
intuitive tool for image analysis applications. It combines indeed a community website for
contributing and sharing tools and material, and software with a high-end visual programming
framework for seamless development of sophisticated imaging work flows. Icy features more than 150
applicative plugins covering such diverse tasks as image enhancement, filtering, active contours,
cell segmentation and tracking, particle detection and tracking. Icy is available at
http://icy.bioimageanalysis.org/.
References 1. Pop, S., Dufour, A., Le Garrec, J.-F., Ragni, C.V., Cimper, C., Meilhac, S.M., and Olivo-Marin, J.-C. (2013)
Extracting 3D cell parameters from dense tissue environments: Application to the development of the mouse heart, Bioinformatics, doi: 10.1093/bioinformatics/btt027
2. Buisson, J., Chenouard, N., Lagache, T., Blisnick, T., Olivo-Marin, J.-C., Bastin, P. (2013) Intraflagellar transport proteins cycle between the flagellum and its base, Journal Cell Science, 126, 1, 327-38 (doi: 10.1242/jcs.117069)
3. de Chaumont F, Dallongeville S, Chenouard N, Hervé N, Pop S, Provoost T, Meas-Yedid V, Pankajakshan P, Lecomte T, Le Montagner Y, Lagache T, Dufour A, and Olivo-Marin, J.-C. (2012) Icy: an open bioimage informatics platform for extended reproducible research, Nature Methods, 9, 7, pp. 690-6
4. de Chaumont, F., Dos-Santos Coura, R., Serreau, P., Cressant, A., Chabout, J., Granon, S. and Olivo-Marin, J.-C. (2012) Computerized video analysis of social interactions between mice, Nature Methods, 9, 4, pp. 410-7
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 12
Sponsored by:
Introduction to image processing and analysis using FIJI/ImageJ
Szymon Stoma
ETH (Swiss Federal Institute of Technology), Zürich, Switzerland
Handling of microscopy images in scientific research requires at least basic understanding of digital
images. A digital image translates to numbers and is hence a quantitative by nature. During the
introductory lecture I will discuss the very basics of the numerical nature of digital images including
image representation, spatial and bit-depth resolution, image dimensions, lookup tables, basic
mathematical operations and convolution-based filters. During the presentation I will introduce
ImageJ, a public domain software package for image processing and analysis developed initially by
Wayne Rasband (http://imagej.net). Many scientists especially biologists use ImageJ for their research,
so the package became a de facto a standard tool for dealing with images in Biology.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 13
Sponsored by:
Αnalysis of digital images and videos derived from multiphoton microscopy intravital imaging of the neurovascular niche following neuroinflammation
Dimitra Thomaidou
Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, Athens, Greece
The importance of the Neurovascular Niche (NVU) in the maintenance of cerebral homeostasis is
being gradually established during the last years. The term “Neurovascular Unit” (NVU) is used to
describe the integrated system of vascular and neuronal cells and the surrounding glia working in
concert to enable proper brain homeostasis and function. The NVU is affected in many CNS
conditions and plays a role in their pathology. Neuroinflammation is a common feature of many
neurodegenerative and neuroimmunological diseases and if uncontrolled, it can result in the constant
activation of microglia and astrocytes, two of the major components of the NVU. This can generate a
neuroinflammatory environment which can further promote neurodegeneration. Thus, the study of the
dynamic cellular interactions in the pathological NVU could contribute to the identification of
numerous potential targets for treatment in the context of many CNS pathologies. The dynamics of
such processes can be adequately captured by intravital in vivo imaging, which allows the study of
cellular responses to environmental stimuli and cell-cell interactions in the living brain in real time.
Accordingly, in the present study 2-Photon Laser Scanning Microscopy (2P-LSM) has been applied to
follow in real time the morphological changes and interactions of microglia, astroglia and the brain
vasculature under neuroinflammatory conditions induced by the lipopolysaccharide (LPS), which is a
potent activator of systemic inflammation. Analysis of collected imaging data, following intravital
brain imaging using Imaris v.7.4.2 and Icy Bioimage Analysis Software, revealed cellular changes
both in astroglia and microglia upon LPS exposure, characteristic of a strong and long-term
neuroinflammatory response accompanied by quick astroglia and microglia activation. Additionally,
the observed infiltration of inflammatory cells from the periphery inside the brain tissue is considered
to be indicative of a decrease in the blood-brain-barrier (BBB) permeability during these conditions.
Further study will focus on the establishment of BBB leakage in our model and the possible protective
role of the polarized astrocytes under these circumstances.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 14
Sponsored by:
Icy: new features and future!
Fabrice de Chaumont
Bioimage Analysis Unit, Institut Pasteur, Paris, FRANCE
Icy is a free open-source bio-image analysis software. During its 4 years of life it has been
continuously improved and continuously brings new features to users and developers. Today Icy is
used by more than one thousands of regular users which appreciate its intuitive GUI, its ray-traced 3D
visualization and its cutting-edge analysis methods. Users can also adapt and create new algorithms
with script and the graphical programming protocol designer and store them on the Icy website which
centralizes all resources (http://icy.bioimageanalysis.org). Thus it makes available plug-ins, protocols
and scripts to everybody. Centralization also allows searching directly from within the application for
specific features and enables it in a one-click install. During the last months, Icy has received new
exciting features: revisited 3D rendering based on latest VTK version and a new powerful Undo
framework. Icy is now addressing big data: we introduced streaming capability, cluster deployment
and headless calls. Finally, Icy is always evolving, thanks to the feedback of all its users on
http://icy.bioimageanalysis.org/support!
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 15
Sponsored by:
Deciphering Cellular Morphodynamics using Bioimage Informatics
Alexandre Dufour
Bioimage Analysis Unit, Institut Pasteur, Paris, FRANCE
Cell deformation and migration are key factors involved in numerous aspects of cell development,
immune responses, cancer and infectious diseases. Our team develops image analysis tools and
software to extract quantitative information from multi-dimensional, multi-modal live microscopy,
with the aim to derive mathematical models of cellular morpho-dynamics. Throughout this talk, I shall
describe some of our algorithmic developments to a) automatically track multiple cells in 2D/3D
microscopy in cluttered environments and low signal-to-noise conditions, b) efficiently describe the
3D morphology of cells despite their natural shape variability, and c) extract biophysical quantities at
the intra-cellular level directly from imaging data. Finally, we shall emphasize the need for open-
source tools and software by presenting the Icy platform (http://icy.bioimageanalysis.org), a next-
generation software dedicated to the bioimaging communities by providing the latest developments in
image acquisition, visualisation, analysis and computational modeling.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 16
Sponsored by:
Imaging Prions spreading in neurodegenerative diseases: Role of Tunneling
Nanotubes (TNTs)
Chiara Zurzolo
Membrane Traffic and Pathogenesis Unit, Pasteur Institute, Paris France
Neurodegenerative diseases (NDs) such as Prion disease, Alzheimer’s disease (AD), Parkinson’s
disease (PD) and Huntington’s disease (HD) are part of a larger group of protein misfolding disorders
(PMDs) characterized by the progressive accumulation and spreading of protein aggregates of
different sizes - oligomers, protofibrils or fibrils-, which ultimately can assemble into extracellular
amyloid deposits and/or intracellular inclusions. The best-characterized example of PMDs is prion
diseases, which are caused by the conversion of the normal form of the prion protein (PrPC), to a
misfolded form (PrPSc) through “template conformation changes”. Like in Prion diseases, misfolded
forms of ASYN, tau, Abeta and Htt proteins associated with AD, PD and HD can be transmitted
experimentally in cellular and in animal models where it can act as ‘seeds’ to recruit the endogenous
protein into aggregates (seeding process). However, the mechanism of intercellular transfer is still
obscure.
We have recently described a novel mechanism of PrPSc transmission through Tunneling Nanotubes
(TNTs). TNTs are actin-based fine protrusions connecting sparse cells in culture and represents a
novel mechanism of cell-to-cell communication. By using live and quantitative imaging we have
analysed TNT formation and their role in prion transfer. We found that TNT are mediating both
exogenous and endogenous PrPSc transfer between infected and naïve mouse neuronal cells and
between bone-marrow dendritic cells and primary neurons. Furthermore, mutant polyQ Htt aggregates
appear to highjack TNTs as well as fibrillar and oligomeric ASYN assemblies. We propose that TNTs
might contribute to the progression of the pathology of neurodegenerative diseases associated with the
spreading in the brain of misfolded protein assemblies. I will discuss our novel data mainly based on
imaging techniques on the mechanism of TNT formation and their role in the spreading of protein
aggregates involved in neurodegenerative diseases.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 17
Sponsored by:
Imaging phagosome formation, closure and maturation in normal and
virus-infected macrophages
Florence Niedergang
Institut Cochin (Inserm U1016, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité, Paris, France
Phagocytosis and degradation of microorganisms or debris by macrophages is crucial for
pathogen clearance and resolution of inflammation1. The internalization step of phagocytosis relies
on a profound remodeling of the plasma membrane and the cytoskeleton. The local, intense and
transient actin polymerization is the force that deforms the plasma membrane to form pseudopodia1,2.
We set up an original experimental approach, based on Total Internal Reflection Fluorescence (TIRF)
Microscopy to monitor the spatio-temporal reorganization of membrane and actin in nascent
phagosomes in three dimensions in living macrophages3. We observed that vesicular delivery occurs at
the base of the phagocytic cup where actin is depolymerized, while F-actin is present in extending
pseudopods. We showed that the NF-kB-signaling protein B cell lymphoma/leukemia-10 (Bcl10) is
unexpectedly involved in actin remodeling during phagocytosis by controlling the traffic of
intracellular vesicles bearing the AP1 adaptors and the OCRL phosphatase4. Using this system, we
also revealed that a crosstalk between actin and dynamin-2 takes place during phagosome formation,
and that dynamin-2 plays a critical role in the effective scission of phagosomes from the plasma
membrane5.
Macrophages are a main target and reservoir for the Human Immunodeficiency Virus (HIV)-1. HIV-
infected macrophages exhibit defective functions that contribute to the development of opportunistic
diseases. We demonstrated that macrophages infected with HIV-1 exhibit defects in phagocytosis,
which are due to the major virulence factor Nef, perturbing focal exocytosis of intracellular
compartments6. More recently, we identified the viral factor Vpr as a modulator of the microtubule
dependent endocytic trafficking that alters localization of microtubule binding proteins EB1 and
p150Glued, leading to strong alterations in phagosome movement and phagolysosome biogenesis7. To
measure the speed of centripetal movement of phagosomes in individual HIV-GFP-infected
macrophages, we used a combination of bright field and fluorescence confocal microscopy8. We also
used correlative electron microscopy to analyze the development of bacteria in HIV-infected
macrophages.
Together, our data aim to understand how opportunistic pathogens develop in HIV-1 infected
macrophages.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 18
Sponsored by:
REFERENCES
1. Niedergang F., Phagocytosis. In: Ralph A Bradshaw and Philip D Stahl (Editors-in-Chief), Encyclopedia of Cell Biology, Vol 2, Waltham, MA: Academic Press, 2016, pp. 751-757. Book chapter 2. Deschamps C, Echard A and Niedergang F. Phagocytosis and cytokinesis : do cells share common tools ? (2013) Traffic 4, 355-364. Review 3. Marie-Anaïs F, Mazzolini J, Bourdoncle P and Niedergang F. (2016). “Phagosome Closure Assay” to visualize phagosome formation in three dimensions using Total Internal Fluorescent Microscopy (TIRFM). JoVE in press. 4. Marion, S., Mazzolini, J., Herit, F., Bourdoncle, P., Kambou-Pene, N., Hailfinger, S., Sachse, M., Ruland, J., Benmerah, A., Echard, A., et al. (2012). The NF-kappaB Signaling Protein Bcl10 Regulates Actin Dynamics by Controlling AP1 and OCRL-Bearing Vesicles. Dev Cell 23, 954-967. 5. Marie-Anaïs F, Mazzolini J, Herit F and Niedergang F. (2016). Dynamin-actin cross-talk contributes to phagosome formation and closure. Traffic 17, 487-499. 6. Mazzolini, J., Herit, F., Bouchet, J., Benmerah, A., Benichou, S., and Niedergang, F. (2010). Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments. Blood 115, 4226-4236. 7. Dumas A., Lê-Bury G., Marie-Anaïs F., Herit F., Mazzolini J., Guilbert T., Bourdoncle P., Russell D.G., Benichou S., Zahraoui A., and Niedergang F. (2015). The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking. J. Cell Biol. 211 : 359-372. 8. Lê-Bury G., Deschamps C., Dumas A. and Niedergang F. Phagosome migration and velocity measured in live primary human macrophages infected with HIV-1. JoVE in press.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 19
Sponsored by:
Morphometric analysis of neuronal networks: from memory to super-
resolution structure of the synapse
Lydia Danglot
Institut Jacques Monod, Université Paris Diderot, Inserm 950 "Membrane trafic in Health & Disease", France
http://lydia.danglot.free.fr Keywords: synapse, plasticity, super-resolution microscopy, SIM, STORM, brain tile imaging. The nervous system contains billions of neurons that communicate with each others via specialized junctions: the synapses. Synaptic dysfunction is at the heart of the etiology of a wide range of neurological and psychiatric disorders (Alzheimer's disease, autism spectrum disorders or schizophrenia). Indeed, many diseases are characterized by altered distribution and / or mis-apposition of the pre and post-synaptic sites. We propose here to describe imaging techniques and image analysis tools to detect, count and characterize such defects through spatial statistics. We will describe imaging techniques used to study from neuronal network, to single molecule localization microscopy. These wide technical approaches will be described through the characterization of our biological models in the lab which is the characterization of transgenic mice line knocked out for a vesicular SNARE protein involved in fusion process.
Indeed, the establishment, maintenance and plasticity of the synapse require the transport, recycling and degradation of membrane-associated synaptic molecules via membrane trafficking mechanisms. Vesicular and target-SNAREs play a central role in all membrane trafficking pathways. Neurons express several v-SNAREs including TI-VAMP/VAMP7 which is involved in Golgi to plasma membrane transport particularly of membrane micro domains, pre synaptic exocytosis, lysosomal secretion and autophagosome biogenesis and secretion. We previously found that TI-VAMP/VAMP7 KO mice are viable and characterized by decreased brain weight, increased ventricle volume as measured by MRI, and increased anxiety suggesting a role for VAMP7 in higher brain functions [1]. In addition, VAMP7 was previously found to be particularly enriched in the hippocampus, a brain structure involved in learning and memory, its expression being both pre- and post-synaptic.
Here we found that VAMP7 KO mice showed increased memory performance and a decreased memory decline in old animals compared to wild-type. We are now deciphering at the tissular, cellular and molecular level what could explain such increased performances.
We investigated the neuronal morphology in situ by tile imaging of the entire brain (Golgistaining) to realized mosaic image at a good resolution in order to discriminate dendritic arbors. Those results indicated profuse dendritic pyramidal arborization in specific regions of the KO brain suggesting altered morphological and potentially functional post-synaptic properties. This was further confirmed by electron microscopy which unraveled both pre and post-synaptic structural modifications in the KO. Neuronal network and spine density is actually approached by tile confocal imaging and 3D reconstruction. At the synapse level, we found altered synaptic biochemical content in glutamatergic receptors and associated molecules by biochemical approaches. Determining the precise localization of molecules using image based analysis is a standard and powerful tool to probe molecular assembly in cellular compartments. We further investigated the localization of the synaptic molecule by multicolor super-resolution microscopy (SIM and STORM). Principle and imaging technics will be presented. We are
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 20
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currently developing image analysis software in collaboration with T. Lagache and JC. Olivo Marin team (Institut Pasteur) to analyze precise distance between coupled synaptic molecules by robust statistical approaches [2]. While standard colocalization techniques have been widely used in standard fluorescence microscopy, the emergence of super resolution microscopy made obsolete the classical overlap approaches and calls for new methods of analysis. We developed a method and software (SODA: Statistical Object Distance Analysis) that statistically quantifies the relative spatial positioning of several molecules' populations. The method computes the coupling distance and specifically quantifies the morphology (size, shape, intensity) of coupled spots. We will present the example of the apposition of three consensual synaptic molecules in primary hippocampal neurons with structured-illumination microscopy and STORM microscopy. We will present advantage of both methods and explain why they are complementary.
REFERENCES
1. Danglot L, Zylbersztejn K, Petkovic M, Gauberti M, Meziane H, Combe R, Champy MF, Birling MC, Pavlovic G, Bizot JC, Trovero F, Della Ragione F, Proux-Gillardeaux V, Sorg T, Vivien D, D'Esposito M, Galli T. J Neurosci. 2012 Feb 8;32(6):1962-8.
2. Danglot L, Freret T, Le Roux N, Narboux Nême N, Burgo A, Hyenne V, Roumier A, Contremoulins V, Dauphin F, Bizot JC, Vodjdani G, Gaspar P, Boulouard M, Poncer JC, Galli T, Simmler MC. J Neurosci. 2012 Jun 27;32(26):9007-22.
3. T. Lagache, N. Sauvanet, L. Danglot and JC. Olivo-Marin, “Statistical analysis of molecule colocalization in bioimaging.”Cytometry par A. 2015, vol. 87, no. 6, pp. 568–79, 2015.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
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Systematic analysis of the secretory pathway in mammalian cells
Franck Perez
Institut Curie, CNRS, Paris, France
It is now clear that multiple routes exist in cells to transport proteins and lipids toward various
compartments. To analyze these routes, there is a strong need for a versatile, highly synchronized
assay that can be used at physiological temperature, in living cells. We developed the RUSH system
(Retention Using Selective Hooks), a two-state assay based on the reversible interaction of a Reporter
protein with a Hook protein stably localized in a donor compartment. This system can be used to study
and quantify the trafficking of very diverse proteins of different size, shape and destined to various
compartments. The RUSH system can be used for real time monitoring of transport in living cells as
well as for High Throughput screening using automated imaging.
We are using the RUSH assay at multiple scales to monitor the transport of proteins in cells and
analyze their intracellular signatures, to map the diversity of trafficking routes in terms of biochemical
composition, to integrate these routes with other cellular machineries, like cellular adhesion and
polarization, and to screen diverse chemical libraries and identify potential therapeutic molecules.
During this lecture, I will present data obtained using spinning disk microscopy, TIRF microscopy and
High Content imaging. Data obtained using traction force microscopy and cells grown on patterns will
also be shown. Analysis of these data was performed using FIJI/ImageJ and the In Cell Analyzer
software.
REFERENCES
1. Boncompain, G., Divoux, S., Gareil, N., de Forges, H., Lescure, A., Latreche, L., Mercanti, V., Jollivet, F., Raposo, G., and Perez, F. (2012). Synchronization of secretory protein traffic in populations of cells. Nat Methods 9, 493-498.
2. Boncompain, G., and Perez, F. (2013). Fluorescence-based analysis of trafficking in mammalian cells. In Methods Cell Biol, pp. 179-194.
3. Terai, T., Kohno, M., Boncompain, G., Sugiyama, S., Saito, N., Fujikake, R., Ueno, T., Komatsu, T., Hanaoka, K., Okabe, T., et al. (2015). Artificial Ligands of Streptavidin (ALiS): Discovery, Characterization, and Application for Reversible Control of Intracellular Protein Transport. J Am Chem Soc 137, 10464-10467.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 22
Sponsored by:
Automated High Throughput Microscopy-based RNAi screening
of mammalian cell invasion by the bacterial pathogen Listeria monocytogenes
Javier PIZARRO-CERDA
Systems Biology of Bacterial Infections Group, Institut Pasteur, Paris, France
The gram-positive bacterium Listeria monocytogenes is the causative agent of human listeriosis, a
food-borne disease characterized by gastroenteritis, meningitis and abortions. A crucial stage in
infection is the bacterial internalization and intracellular replication process in phagocytic and
normally non-phagocytic cells. Indeed, Listeria is able to promote its entry within mammalian cells, to
disrupt its internalization vacuole, to proliferate in the host cell cytoplasm and to use an actin-based
motility system to move within cells and to spread to neighboring cells (Pizarro-Cerda et al. 2012). We
have recently designed high-throughput microscopy-based screens in order to: a) investigate late
cellular infection, and b) discriminate between bacterial entry events and escape from the
internalization vacuole.
To investigate late cellular infection, we established an assay relying on the cytosolic detection of
InlC, a bacterial protein from the internalin family that is highly expressed and secreted by
intracellular Listeria. By using antibodies to label InlC, we are able to differentiate infected from non-
infected cells (Kühbacher et al. 2013). We used therefore this microscopic assay to investigate cellular
pathways required for Listeria infection using an experiment protocol that includes: a) the reverse
RNAi transfection of HeLa cells in 384-well plates for 72 hours using diverse human RNAi libraries,
b) the infection of transfected cellular monolayers with a L. monocytogenes EGDe.PrfA* strain
expressing the enhanced-GFP for 1 hour, c) the killing of non-invading bacteria with a gentamicin
solution for 4 hours, d) the fixation/permeabilization of cells and e) their labeling with DAPI for
nuclear staining, with fluorescent phalloidin for actin staining and for InlC using polyclonal anti-InlC
antibodies to specifically identify L. monocytogenes infected cells. Using the public software
CellProfiler (http://cellprofiler.org/) (Broad Institute), we then: I) used a ‘Mixture of Gaussian’ (MoG)
thresholding method to segment the nuclear DAPI staining and identify the number of cells per image,
II) used a ‘Propagation/Otsu Global’ thresholding method to identify the cytoplasm of cells using the
actin staining, and III) classified ‘infected’ versus ‘non-infected’ cells using the signal from the InlC
labeling. Infection indexes were estimated by dividing the number of infected cells by the total number
of cells within a specific monolayer for a specific RNAi treatment, and a Z-score is calculated after
logarithmic transformation of the infection indexes -which permits the comparison among results from
plates generated in different experiments (Rämö et al. 2014). Using this approach, we identified the
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 23
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existence of different Arp2/3 complexes in cells which differentially regulate actin polymerization at
Listeria entry sites and during cytoplasmic actin-based motility (Kühbacher et al. 2015).
To discriminate between bacterial entry events and escape from the internalization vacuole, we
generated a Listeria strain which expresses in its surface a β-lactamase, which allows cleavage of the
fluorescent probe CCF4. In cells loaded in their cytoplasm with CCF4, bacteria which can not escape
their phagosome can not have access to the probe, which upon excitation will emits a FRET
fluorescent signal at 520 nm. However, when bacteria are able to escape their phagosomes, their
surface β-lactamase will cleave the CCF4 which will inhibit FRET and display fluorescence at 447
nm. Using an Opera Imaging System coupled with the Acapella software (PerkinElmer) we first
analyzed the FRET signal to identify cells in which Listeria vacuolar escape took place. In the same
samples, we then performed a differential bacterial staining (Kühbacher et al. 2014) to differentiate
extracellular from total bacterial populations, and again using the Opera Imaging System/Acapella
software we estimated in the proportion of bacteria which invaded cells (Quereda et al. 2016),
discriminating entry from vacuolar escape.
REFERENCES
1. Kühbacher et al. 2013. J Vis Exp 19: e51043. 2. Kühbacher et al. 2014. Methods Mol Biol 1157: 167. 3. Kühbacher et al. 2015. mBio 6: e00598-15. 4. Pizarro-Cerda et al. 2012. Cold Spring Harb Perspect Med 2: a010009. 5. Quereda et al. 2016. Appl Environ Microbiol 82: 211. 6. Rämö et al. 2014. BMC Genomics 15: 1162.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 24
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Understanding malaria parasite migration
Freddy Frischknecht
Center for Infectious Diseases, Heidelberg University, Heidelberg, Germany
Malaria parasites are transmitted by mosquitoes, which inject the parasites into the skin of the host.
There, parasites migrate at extremely high speeds penetrating the tissue and searching for a blood
vessel that they need to enter to continue their journey. We aim at understanding the rapid motility.
Like eukaryotic motility it depends on an interaction of actin and myosin as well as substrate contact.
However, unlike most eukaryotic motility malaria parasites do not change their shape during
migration. I will present our mixed approach of generating parasite lines expressing fluorescent or
altered actin binding proteins and how we investigate these lines using a range of biophysical assays
and micro-structured or nano-patterned substrates.
These assays include the use of (i) soft substrates with embedded fluorescent microspheres to
investigate traction forces generated by the parasites (Munter 2009); (ii) the use of poly-
dimethylsiloxan pillar arrays as structured micro-environments to investigate migration patterns of
parasites (Hellmann 2011); (iii) the use of optical tweezers to investigate the membrane flow of
parasites as well as force generation on the parasite surface (Quadt 2016); the use of tiny gold beads
for generation of nano-structured surfaces to investigate the numbers of surface proteins needed for
substrate adhesion and their influence on motlity (Perschmann 2011). We utilize standard image
analysis routines based on ImageJ as well as custom written plugins.
During the afternoon practical sessions, I will be discussing some examples of bad image
arrangements for figure generation in the hope to stimulate students to only generate perfect figures in
their theses and papers.
REFERENCES
1. Quadt KA, Streichfuss M, Moreau CA, Spatz JP and Frischknecht F, Coupling of retrograde flow to force production during malaria parasite migration, ACS Nano 2016; 10: 2091-2102.
2. Perschmann N, Hellmann JK, Frischknecht F and Spatz JP, Induction of malaria parasite migraion by synthetically tunable microenvironments. Nano Letters, 2011; 11: 4468-4474.
3. Hellmann JK, Münter S, Kudryashev M, Schulz S, Heiss K, Müller AK, Matuschewski K, Spatz JP, Schwarz US and Frischknecht F, Environmental constraints guide migration of malaria parasites during transmission. PLoS Pathogens, 2011; 7: e1002080.
4. Münter S, Sabass B, Selhuber-Unkel C, Kudryashev M, Hegge S, Spatz JP, Engel U, Matuschewski K, Schwarz US and Frischknecht F, Plasmodium sporozoite motility is modulated by the turnover of discrete adhesion sites. Cell Host & Microbe 2009; 6: 551-562.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 25
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Multi-Imaging Approaches to Decode How Toxoplasma Parasites
Invade Target Cells
Isabelle Tardieux
Institut of Advanced BioSciences, INSERM U1209, CNRS UMR5309, Univ. Grenoble Alpes, Team Membrane Dynamics of Host-Parasite Interactions, [email protected] Apicomplexa constitute a large phylum of protozoan parasites, which includes medically important
human pathogens such as Toxoplasma and Plasmodium, the agents of toxoplasmosis and malaria
respectively. Occurring within the context of immunocompromised human beings, the public health
threats imposed by Toxoplasma are directly associated with the ability of the transient extracellular
Toxoplasma crescent shaped stage, called tachyzoites (7 to 3 μm length/width), to invade and expand
its population size, inside most non phagocytic and phagocytic cells. Host cell invasion, a unique and
spectacularly rapid process (lasting few seconds) is arguably one of the most scrutinized scientific
questions in Apicomplexan biology with the relevant perspective of designing anti-invasive molecules
to prevent disease. A shared multi-step model of host cell invasion by Apicomplexan parasites has
been proposed about 30 years ago, with the parasite establishing a tight junction with the host cell
plasma membrane and then actively penetrating into a nascent parasitophorous vacuole. Of note,
recent datasets generated with both genetically engineered Plasmodium and Toxoplasma parasites and
host cells have cast doubt on and revealed flaws in this model, the major highly debated questions
being:
1- the force contribution of each partner during invasion.
2- the role of the host cell cortex to control junction shaping and function during parasite entry.
We will discuss imaging approaches we have undertaken to address these issues that include:
- Fluorescent Speckle Microscopy to follow actin flow and turnover in live cells following
microinjection of fluorescent actin. It relies on the use of conventional wide-field fluorescence light
microscopy, on digital imaging with a low-noise CDD camera and on Matlab image acquisition and
analysis.
- Fluorescent confocal real time video-microscopy of invasion events with multi-channel image
acquisition using a spinning disk and a coolsnap HQ2 camera driven by the Metamorph software.
Image processing includes trajectory analysis and allows kinematic modeling (Matlab) to monitor
Toxoplasma pre-invasive and invasive behavior. These approaches have also allowed monitoring
simultaneously host cell actin and membrane dynamics during parasite invasion.
- Automatic image capture and data analysis for quantitative Toxoplasma invasion assays
performed on 96 well plates and simultaneously under various settings using the Olympus Scan^R
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
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automated inverted microscope and Scan^R software (object and sub-object detection, parameter
calculation, specific gating and classification schemes).
- Force measurement using Atomic Force Microscopy coupled to fluorescent imaging to
attempt getting direct insights on the force contribution of tachyzoites during attachment/ invasion of
host cells and on the changes of host cell cortex tension during these events.
These imaging techniques have been proved decisive to properly analyze Toxoplasma phenotypes for
wild type and “invasion-related” mutants. These data also provide new tools and concepts to study
membrane and cortex tension of mammalian cells, a topic of increased relevance to understand cell
motility, division and features of post mitotic cells.
REFERENCES
1. Delorme-Walker V, Abrivard M, Lagal V, Anderson K, Perazzi A, Gonzalez V, Page C, Chauvet J, Ochoa W, Volkmann N, Hanein D, Tardieux I. (2012). Toxofilin upregulates the host cortical actin cytoskeleton dynamics, facilitating Toxoplasma invasion. J Cell Sci.;125 :4333-42
2. Bichet M, Joly C, Henni A, Guilbert T, Xémard M, Tafani V, Lagal V, Charras G, TARDIEUX I. (2014). The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force. BMC Biol.;12:773
3. Marion Bichet, Bastien Touquet, Virginie Gonzalez, Markus Meissner Isabelle Tardieux (2016). Toxoplasma relies upon high-speed myosin motor to restrict host cell membrane dynamics and compressive forces during invasion- eLife submission.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
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Sponsored by:
Image ethics for publication
Jerome Mutterer
CNRS-IBMP, 12, rue du general Zimmer, 67084 Strasbourg, France
Microscopy images carry a considerable amount of data. The lectures and practical trainings provided
in this course aim at presenting state of the art methods to obtain usable images and extract significant
data from them. Another step forward is publishing those images. The publication step is of major
importance as the published data is expected to guarantee both a perennial link to the original raw data
and sufficient information for research reproducibility. I’ll discuss common pitfalls associated with
publishing image data and present existing tools that can be used by scientists. On one side I will
present solutions for mandatory storage of original raw data, that range from laboratory or institution
secured storage to scientific online data stores, in a context where the amount of acquired raw data
itself becomes challenging. On the other side, I’ll suggest and demonstrate the use of available tools
for securing reproducible image processing workflows, as available in different image acquisition or
image analysis software.
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 28
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ImageJ/Fiji hands on practical session
Szymon Stoma
ETH, Zürich, Switzerland
The goal of these workshop sessions is to introduce the most important concepts of working with
images in biology, while teaching you how to use some very popular image analysis software. To
achieve this goal I will go over various usages of FIJI/ImageJ. Learning to use FIJI/ImageJ properly
often enables people to understand quantitative aspects of image processing and analysis. We start
with GUI usage, and then extend this to writing ImageJ macros*. Images are everywhere in science, so
if you continue in this area you are quite likely to encounter them at some point - perhaps directly
through your own need to perform analysis, in helping colleagues, or in evaluating whether some
analysis reported in a paper has been done sensibly.
Part 1 Understanding images and FIJI/ImageJ basics
Part 2 Handling dimensions (2D+ images) & measurements
Part 3 Pixel processing & thresholds
Part 4 Segmentation, filters & transforms
Part 5 Towards quantitative image analysis
(Part 6 Automating analysis)*
Monday 4 July: Part 1-3
• Image examination (bit depths, saturation, offset, dynamic range, LUT)
• Comparison of different image formats/ 8-bit conversion
• Color images
• Image formats
• Image restoration
• Measurements
o Intensity measurements in multiple regions
o Biological interpretation of intensity measurements
• Thresholding and particle analysis
• Multidimensional images
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 29
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Wednesday 6 July: Part 4-6*
• Mathematical operations
• Image filtering
• Smoothing to detect objects in noisy images
• Segmentation
• Background subtraction on an image
• (Macros)*
o Reproducibility
o Automation
o Workflow optimization
*) depending on group tempo, interactions with ICY, etc.
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Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 30
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Icy hands on practical session
Fabrice de Chaumont, Stéphane Dallongeville, Alexandre Dufour
Bioimage Analysis Unit, Institut Pasteur We present Icy, an open source and free software dedicated to the bio-image analysis. Icy provides an
integrated platform that aims at bridging the gap between developers and users, by combining: a) an
open-source image analysis software, offering a powerful and flexible environment for developers
such as applied mathematicians to write algorithms fast and efficiently; b) a common set of tools to
view and manipulate data, and a set of plugins to perform specific quantification or analysis on
images; c) a community-based website centralizing all plugins and resources to facilitate their
management and maximize their visibility towards users.
During the course, we will focus on: a) using the software for analysis and data visualization purposes;
b) understand the file format, effect of compression over images on the noise data, and how much
noise data is important in the image analysis; c) the use of look up table and false color representation;
d) multiple use of ROI and their edition; e) creating ROI automatically through segmentation plugins;
f) detecting spots and performing tracking analysis; g) using batch processing.
Then we will focus on the autonomy Icy provides to the biologists by using its graphical programming
and scripting capabilities through various examples.
We will then finish by demonstrating how to publish original work created during this workshop.
All along the course, participants will be invited to use their own images, and apply the “Icy” methods
directly on their analysis.
Tuesday 5 July
Icy free software http://icy.bioimageanalysis.org/
• Installing the software
• Exploring the image with a 2D visualization
• Using the look up table, and understanding the dynamic of the image
• Explain image acquisition to avoid the loss of information
• Understanding the noise in an image, and why its evaluation is needed
• Using the Region of Interest
• Using a ray-tracer in 3D to visualize the images.
• Practical example: an analysis of an atomic force microscopy acquisition
• Browsing for functionalities and Installing a plugin
• Detecting spots in 2D/3D or 4D
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• From the quantitative result to biological deduction: good practice and traps
• Performing a tracking and analyzing the results
• Tracking of big objects with deformable models
• Track objects over time in 2D and 3D
• Generate object statistics on length, speed, duration and distance from origin, displacement
straightness etc.
Thursday 7 July
• General introduction to co-localization analysis
• Correlation and Overlap Based methods
• Pearson and Mander’s Coefficient
• Statistical significance and quantification
• Object-based methods
• Molecule detection and spatial analysis
• Statistical significance and quantification
• Co localization Studio and Co localization Simulator
• Impact of noise on the different methods
• Test with synthetic images
• Perspectives: 3D, time and non-parametric methods
• Creating complex analysis workflow and batch processing with graphical programming and
scripting
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ADDRESSES
of
Instructors
and
Participants
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Teaching Team
Name Institution Country E-mail
Haralabia Boleti
Light Microscopy Unit, Hellenic Pasteur Institute
http://www.pasteur.gr/?page_id=244&lang=en
HELLAS
Fabrice de Chaumont
Institut Pasteur, Paris , Bioimage Analysis Group
http://icy.bioimageanalysis.org/
FRANCE [email protected]
Stephane Dallongeville
Institut Pasteur, Paris , Bioimage Analysis Group
http://icy.bioimageanalysis.org/
FRANCE
Alexandre Dufour
Institut Pasteur Paris,
Bioimage Analysis Group
http://www.bioimageanalysis.org/member/alexandre-dufour/
FRANCE
Lydia Danglot
Institut Jacques Monod,Université Paris Diderot, Inserm 950 "Membrane trafic in Heath & Disease"
http://lydia.danglot.free.fr
FRANCE
Freddy Frischnecht
Center for Infectious Diseases, Heidelberg University
http://www.klinikum.uni-heidelberg.de/Malaria-3-Frischknecht.100117.0.html
GERMANY
Jean-Christophe Olivo-Marin
Institut Pasteur, Paris
Bioimage Analysis Group
http://www.bioimageanalysis.org/index.html
http://icy.bioimageanalysis.org/
FRANCE
Jerome Mutterer Institut de Biologie Moléculaire des Plantes, CNRS UPR2357, Strasbourg
http://ibmp.u-strasbg.fr/
FRANCE
Florence Niedergang
Team « Biology of Phagocytes », Institut Cochin, Inserm U1016-CNRS UMR8104- Université Paris Descartes, Paris
http://NiedergangLab.free.fr
FRANCE
Instructors’ Team
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http://cochin.inserm.fr/Departements/ih/equipe-niedergang/
Franck Perez
Team of Dynamics of Intra-Cellular Organization, Institut Curie, Paris
http://umr144.curie.fr/en/profile/franck-perez-001079
FRANCE
Javier Pizarro-Cerda
Institut Pasteur, Paris,
Systems Biology of Bacterial Infections,
https://research.pasteur.fr/en/member/javier-pizarro-cerda/
FRANCE
Szymon Stoma
ETH (Swiss Federal Institute of Technology), Zürich, ScopeM
http://stoma.name/pmwiki.php?n=Home.Home
SWITZER
LAND
Isabelle Tardieux
Institut of Advanced BioSciences, INSERM U1209, CNRS UMR5309, Univ. Grenoble Alpes,
Team Membrane Dynamics of Host-Parasite Interactions
FRANCE
Dimitra Thomaidou
Light Microscopy Unit, Hellenic Pasteur Institute http://www.pasteur.gr/?page_id=2444&lang=en
HELLAS
Evangelia Xingi
Light Microscopy Unit, Hellenic Pasteur Institute
http://www.pasteur.gr/?page_id=2444&lang=en
HELLAS
Chiara Zurzolo
Membrane Traffic and Pathogenesis Unit, Institut Pasteur, Paris http://www.pasteur.fr/en/research/cell-biology-infection/units-groups/membrane-traffic-and-pathogenesis
FRANCE
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Sponsored by:
Name Institution Country/ Nationality
Andreadaki Maria IMBB-FORTH HELLAS
Borisova Dayana Institute of Microbiology, Bulgarian Academy of Sciences
BULGARIA [email protected]
Charalambous Maria HPI/U of Thessaly HELLAS/
CYPRUS
Frakolaki Efseveia HPI HELLAS
Hajar Chihab Faculty of sciences Ben M’sik - Casablanca /HPI
MORROCO [email protected]
Herit Floriane Cochin Institute FRANCE
Javed Numan IP Shanghai CHINA/
PAKISTAN
Jung Nicolas University of Luxembourg
LUXEMBURG [email protected]
Kirschmann Moritz Alexander
Institute of Neuroinformatics, ETH Zurich
SWITZERLAND/
GERMANY
Martel Daniel Institut Pasteur, Paris FRANCE
Martzoukou Olga Faculty of Biology, U of Athens
HELLAS [email protected]
Neofytou Konstantina University of Cyprus CYPRUS
Panagi Myrofora University of Cyprus CYPRUS
Panayidou Stavria University of Cyprus CYPRUS
Paunova-Krasteva Tsvetelina
Institute of Microbiology – BAS/RIIP
BULGARIA [email protected]
Participants
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Sponsored by:
Rabiaa Manel Sghaier Institut Pasteur de Tunis
TUNISIA [email protected]
Sinigaglia Laura Institut Pasteur, Paris FRANCE/
ITALY
Paneque Guerrero Taimi Emelia
Center for Genetic Engineering & Biotechnology
CUBA
Tamamouna Vasilia University of Cyprus
CYPRUS
Teloni Savvas University of Cyprus CYPRUS [email protected]
Thanou Irini HPI HELLAS
Tsalavoutas-Psarras Lefteris
Democritus Research center
HELLAS [email protected]
Tziouvara Olympia-Paraskevi
HPI HELLAS [email protected]
Zygogianni Ourania HPI
HELLAS [email protected]
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Sponsored by:
NOTES
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 38
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 39
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 40
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 41
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 42
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 43
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 44
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 45
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 46
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 47
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 48
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 49
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 50
Sponsored by:
2016 RIIP International Course - “Cell Biology and infection: Digital Image Processing/Analysis
Hellenic Pasteur Institute Tools for Quantitative Light Microscopy Imaging” 51
Sponsored by: